Hsv viral vector

ABSTRACT

The present invention relates to a recombinant herpes simplex virus (HSV) viral vector genome which has substantially lost its transducing properties as a result of a DNA sequence change in the gene coding for Vmw65 protein and also comprises an expressable heterologous gene inserted into a region of the HSV genome which is non-essential for the culture of the virus, the gene being under the control of the immediate early (IE1) gene enhancer of cytomegalovirus (CMV) and to the use of the recombinant HSV genome in therapy and vaccination.

FIELD OF THE INVENTION

[0001] The present invention relates to a recombinant herpes simplexvirus (HSV), especially type 1 (HSV-l) or type 2 (HSV-2) having a goodability to continuously express an inserted heterologous gene whilst thevirus is at the same time maintained in its latent non-replicativestate.

BACKGROUND OF THE INVENTION

[0002] A distinguishing feature of herpes virus infections is theability to persist in the host for long periods in a non-replicative orlatent state. Herpes simplex virus type 1 (HSV-1) establishes latentinfection in human peripheral sensory ganglia and can reactivate toproduce recurrent mucocutaneous lesions. Operationally, the pathogenesisof herpes virus infections can be divided into several distinct stageswhich can be studied individually in experimental animal models: acuteviral replication, establishment of latency, maintenance, andreactivation. Following inoculation, HSV-1 replicates at the site ofinoculation and is transported to sensory ganglia. Replication at theperiphery or in sensory ganglia may increase the amount of virus thatcan establish latent infection. During latent infection, HSV-1 DNA canbe detected in infected tissues but infectious virus cannot be detected.This latent state is often maintained for the life of the host. Avariety of stimulae (such as fibrile illness and exposure to ultravioletirradiation) can interrupt the latent state and cause the reappearanceof infectious virus or reactivation.

[0003] Transcription of the HSV-1 immediate early (IE) genes is notdetectable during latency. However, in tissue culture, IE geneexpression is a pre-requisite for viral replication. Transcription ofthe IE genes is transinduced by a virion protein Vmw65 (transinducingfactor) that is a component of the HSV-1 virion. Vmw65 does not binddirectly to HSV-l DNA but mediates transinduction by association withcellular proteins to form a complex which interacts with the IEregulatory element.

[0004] Ace et al (1989) report an HSV-l mutant which contains a 12bpinsertion in the coding region of Vmw65 which is unable to transinduceIE gene expression, though the altered Vmw65 is incorporated into maturevirions.

[0005] The inventor's previous patent specification WO91/02788 disclosesa herpes simplex virus type 1 mutant which includes the mutation withinthe Vmw65 gene which removes the transinducing properties of the Vmw65transactivator protein such that the virus remains in its latent state.In addition, a β-galactosidase marker gene under the control of thelatency associated transcript (LAT) promoter is inserted into thethymidine kinase (TK) gene and expression of the heterologous geneduring latency is observed.

[0006] It is an object of the present invention to provide an HSV viralvector having enhanced expression of the inserted heterologous geneduring latency.

SUMMARY OF THE INVENTION

[0007] Generally speaking, the present invention is based on thediscovery that enhanced long term expression during latency may beobtained by use of the IE1 gene enhancer of cytomegalovirus controllingthe inserted heterologous gene.

[0008] Most specifically, the present invention provides a recombinantherpes simplex virus (HSV) viral vector genome which comprises;

[0009] (i) a DNA sequence change in the gene coding for Vmw65 proteinsuch as to substantially remove transinducing properties thereof; and

[0010] (ii) an expressable heterologous gene inserted into a region ofthe HSV genome which is non-essential for culture of the virus, the genebeing under the control of the immediate early 1 (IE1) gene enhancer ofcytomegalovirus (CMV).

DETAILED DESCRIPTION OF THE INVENTION

[0011] The Vmw65 sequence change removes the transinducing propertiesthereof such that expression of HSV IE genes and therefore HSV viralreplication in vivo, is substantially removed. The HSV vector istherefore constrained to remain in its latent state. The use of the IE1CMV enhancer to control the inserted heterologous gene has been found togive excellent long term expression of the heterologous gene duringlatency. Experiments in mice using the inserted heterologous lacZ genehave showed continuous expression from the latent vector of up to fivemonths. In contrast, use of other promoters such as HSV-1 Vmw110 andVmw65, and the Moloney murine leukaemia virus enhancer have been foundnot to give long term expression during latency.

[0012] The structure of the human cytomegalovirus (HCMV) enhancer isdiscussed in Stinski and Roehr (1985). The IE1 enhancer is thepromoter-regulatory region upstream of the major immediate early gene ofhuman cytomegalovirus. This enhancer region upstream of the IE1 geneconsists of a series of different repeat sequences distributed up to−509bp from the site for the initiation of transcription. Within thisenhancer are a set of inducing sequences. Certain of the sequenceswithin the enhancer region are non-essential and do not effect the levelof expression obtained, whilst other sequences promote downstreamexpression.

[0013] The CMV enhancer is generally that derived from humancytomegalovirus (HCMV) and the immediate early 1 (IE1) nomenclatureapplies particularly to that virus. However, the analogous enhancer fromother types of CMV, such as mouse, rat, equine, simian, and guinea pigCMV, may also be employed.

[0014] The present invention primarily envisages the use of the entireCMV IE1 enhancer sequence. Indeed in a preferred embodiment of theinvention a larger sequence extending to −730bp and including the entireCMV IE1 enhancer was employed. However, it is clearly within the ambitof the skilled man to modify the naturally occurring enhancer sequencewithout departing from the general scope of the present invention. Thus,the present invention is concerned not only with the use of the entireCMV IE1 enhancer sequence but also with variations in that sequence,either by insertion, deletion or substitution such that the enhancerproperties are not substantially affected.

[0015] Other promoter sequences, such as the LAT (latency associatedtranscript) promoter, may be included upstream of the insertedheterologous gene and HMCV enhancer, but these have not been found tooffer any particular advantage according to the present invention.

[0016] The position and size of the DNA sequence change in the genecoding for Vmw65 protein is significant, since it is necessary tosubstantially remove the transinducing properties of the Vmw65 protein(and thereby prevent in vivo replication of the virus and consequentillness of the patient), whilst at the same time retaining thestructural properties of the protein required to successfully assemblethe complete virion when the virus is cultured. The viral vector of thepresent invention must be capable of replication under cultureconditions so as to be able to produce sufficient quantities of themutant virus for use, but at the same time the virus should be incapableof replication in vivo. Preferably, the DNA sequence change is achievedby a transition (purine to purine or pyrimidine to pyrimidine) ortransversion (purine to pyrimidine or vice versa) alteration of 1-72base pairs, an oligonucleotide insert of 3-72 base pairs or a deletionof 3-72 base pairs, at a position between amino acids 289 and 480(especially 289 and 412) of the Vmw65 protein.

[0017] The recombinant HSV may be of type HSV-1 or HSV-2 or may be anintertype recombinant between HSV-1 and HSV-2 which comprises nucleotidesequences derived from both types. The recombinant HSV genome willgenerally be contained in a mutant HSV virus.

[0018] HSV has the ability to infect many tissue types and therefore inprinciple the viral vector of the present invention may be used as avector directed against a wide variety of cell types. Latency in HSVinfection tends to be established within neuronal cells, though it ispossible that expressed gene products may translocate from theiroriginal point of production. The viral vector of the present inventionis thus particularly useful for delivering expressable heterologousgenes into neuronal cells. The genes may deliver a therapeutic effect ormay deliver an antigenic protein for stimulating the production ofantibodies when used as a vaccine. The therapeutic gene is generally agene associated with a neurological genetic deficiency i.e. itcompensates for an inherited or acquired genetic deficiency. Examples ofsuch therapeutic genes include:

[0019] (a) human, rat or mouse tyrosine hydroxylase genes 1, 2 or 3,which are relevant to the alleviation of symptoms of Parkinson'sdisease;

[0020] (b) human, rat or mouse nerve growth factor (e.g. the betasubunit) for treatment of Alzheimer's disease and Parkinson's disease;

[0021] (c) human, rat or mouse hypoxanthine-guanine phosphoribosyltransferase gene for the treatment of Lesch-Nyhan disease;

[0022] (d) human beta-hexosaminidase alpha chain gene, for the treatmentof Tay-Sachs and Sandhoff's diseases; and

[0023] (e) human immunodeficiency virus (HIV) nef gene, for the controlof neurological symptoms in HIV-positive individuals.

[0024] In particular, the in situ expression of tyrosine hydroxylase bythe HSV viral vector of the present invention may help alleviate thesymptoms of Parkinson's disease. Tyrosine hydroxylase is a crucialenzyme in the synthesis of dopamine. Deficiency of dopamine is the majorcause of symptoms in Parkinson's disease, and current treatmentinvolving the administration of L-dopa gives only -short-lived respite.

[0025] The heterologous gene may be inserted into any region of theviral genome which is non-essential for the culture of the virus, i.e.replication of the virus outside the body, particularly in tissueculture. In particular, the insertion of the heterologous gene could bemade in the coding sequences or in the flanking control regions of oneor more of the following HSV-1 genes:

[0026] 1. The thymidine kinase gene (the UL23 gene); which is thepreferred choice since thymidine kinase is important for pathogenicityof HSV, so that deactivation of its gene may reduce potentialpathogenicity of the mutant vector.

[0027] 2. The RL1 gene

[0028] 3. The RL2 gene (otherwise named the IE110 gene)

[0029] 4. The locus encoding the latency associated transcripts

[0030] 5. The UL2 gene (otherwise named the Uracil-DNA glycosylase gene)

[0031] 6. The UL3 gene

[0032] 7. The UL4 gene

[0033] 8. The UL10 gene

[0034] 9. The UL11 gene

[0035] 10. The UL13 gene

[0036] 11. The UL16 gene

[0037] 12. The UL20 gene

[0038] 13. The UL24 gene

[0039] 14. The UL40 gene (otherwise named the gene encoding the smallsubunit of ribonucleotide reductase)

[0040] 15. The UL41 gene (otherwise named the virion host shutoff factorgene)

[0041] 16. The UL43 gene

[0042] 17. The UL44 gene

[0043] 18. The UL45 gene

[0044] 19. The UL46 gene

[0045] 20. The UL47 gene

[0046] 21. The UL50 gene (otherwise named the dUTPase gene)

[0047] 22. The UL55 gene

[0048] 23. The UL56 gene

[0049] 24. The US1 gene (otherwise named the IE68 gene)

[0050] 25. The US2 gene

[0051] 26. The US3 gene (otherwise named the protein kinase gene)

[0052] 27. The US4 gene (otherwise named the glycoprotein G gene)

[0053] 28. The US5 gene

[0054] 29. The US7 gene (otherwise named the glycoprotein I gene)

[0055] 30. The US8 gene (otherwise named the glycoprotein E gene)

[0056] 31. The US9 gene

[0057] 32. The US10 gene

[0058] 33. The US11 gene

[0059] 34. The US12 gene (otherwise named the IE12 gene)

[0060] The UL, US and RL nomenclature system given above is a systematicone, but certain common names of genes are also included.

[0061] Another aspect of the present invention relates to the use of therecombinant HSV viral vector genome comprising an appropriateexpressible therapeutic gene in the therapy of disease, particularlydiseases due to or associated with genetic deficiency. The viral vectormay also be used as a vaccine to deliver an antigenic protein.

[0062] A further aspect of the present invention relates to apharmaceutical composition for administering the viral vector comprisingthe viral vector in admixture with a pharmaceutically acceptablecarrier. Generally, the composition will be formulated for parenteraladministration—usually by injection—in an appropriate acceptable carriersuch as apyrogenic isotonic saline.

[0063] The present invention is hereafter further decribed by way ofexample only, the insertion of a gene (the lacZ gene coding forβ-galactosidase) into the viral vector of the present invention. ThelacZ gene is inserted in order to demonstrate the technology, since thepresence of the gene is easily detectable. However, for therapeutic orother applications, a heterologous gene would be inserted in ananalogous manner; or the lacZ gene could be directly replaced by anotherheterologous gene.

FIGURES

[0064]FIG. 1 is a diagrammatic representation of the plasmids used toconstruct the viruses used in this study. In1853 is a mutant virusaccording to the present invention, whilst the others are for comparisonpurposes. Comparison mutants in1863 and in1891 also contain the HCMVenhancer but these are positive for Vmw65 and TK respectively.

[0065]FIG. 2, titres of viruses after injection into mouse footpads.Virus was assayed in extracts of feet after injection of 8×10⁷ pfu ofin1853 (□), in1863 () or 1×10⁵ pfu of 1814R (∘). The means and range oftitres from four animals are shown. The level of detection was 50 pfuper foot.

EXAMPLES SECTION Materials and Methods

[0066] Cells

[0067] BHK clone 13 cells (Macpherson and Stoker, 1962) were grown inEagle's Medium (Glasgow modification) supplemented with 10% newborn calfserum, 10% tryptose phosphate broth, 100 units/ml penicillin and 100μg/ml streptomycin (ETC₁₀).

[0068] Viruses

[0069] The HSV-1 strain 17 mutant in1814 containing a 12 bp insertion inthe coding sequences for Vmw65, and the rescued ‘revertant’ 1814R, havebeen described previously (Ace et al, 1989).

[0070] To prepare HSV recombinants (summarised in Table 1) containingthe E.coli lacZ gene inserted within the TK gene, plasmid pMJ27 wasfirst constructed. Plasmid pGX166 (kindly supplied by V.G. Preston) isthe cloned HSV-1 strain 17 BamHI p fragment modified by the insertion ofan XhoI linker at the SacI site within the TK coding sequences. TheHindIII site within the pAT153 vector sequences of pGX166 was destroyedby cleavage, end-filling with Klenow enzyme and religation, to yieldpGX166 ΔH3. The E.coli lacZ gene plus simian virus 40 (SV40) promoterand enhancer was excised from plasmid FJ3 (Rixon and McLauchlan, 1990)as a 4073 bp BamHI/XbaI fragment and cloned between the BamHI and XbaIsites of pUC18 (previously modified by insertion of an XhoI linker intothe SmaI site) to yield pUC181acZ. The lacZ gene plus SV40 promoter andplyadenylation signals were excised from pUC181acZ as a SalI/XhoIfragment and cloned into the XhoI site of pGX166 ΔH3. A plasmid in whichthe direction of lacZ transcription was opposite to that of TK wasselected and designated pMJ27. Plasmid pMJ27 contains unique XbaI andHindIII sites flanking the SV40 promoter plus enhancer, and has anunique XhoI site downstream of the SV40 polyadenylation signal.

[0071] The human cytomegalovirus (HCMV) Towne strain enhancer was clonedas a 760 bp Sau3AI fragment (Stinski and Roehr, 1985) from plasmidpHD101-4 (kindly provided by E. Blair) into the BamHI site of pUC18(from which the SphI site had been inactivated by Klenow treatment andreligation), excised as an EcoRI (end-filled)/HindIII fragment andcloned between the XbaI(end-filled) and HindIII sites of pMJ27 in theappropriate orientation, to yield plasmid pMJ101. The HSV-1 Vmw110promoter was excised as an 836 bp BbvI(end-filled)/SacI fragment frompJR3 (Everett, 1984) and cloned between the SphI (Klenow-treated) andSacI sites of pUC18. The promoter was then removed as anEcoRI(end-filled)/HindIII fragment and cloned between theXbaI(end-filled) and HindII1 sites of pMJ27, to yield plasmid pMJ102.The Moloney murine Leukaemia virus (Momulv) promoter plus enhancer wascloned as a 760 bp EcoRI/SmaI fragment, modified by the addition ofBamHI linkers at each end (Lang et al, 1983) into the BamHI site ofpUC18 (without the SphI site, as described above). The Momulv enhancerwas then excised as an EcoRI(end-filled)/HindIII fragment and clonedbetween the XbaI(end-filled) and HindIII sites of pMJ27 to yield pMJ103.The Vmw65 promoter was excised as a 380 bp TaqI/EcoRV fragment from pMC1(Campbell et al, 1984) and cloned between the SphI (Klenow treated) andAccI sites of pUC18. The promoter was excised as a XbaI/HindIII fragmentand cloned between the XbaI and HindIII sites of pMJ27, to yield pMJ104.The structures of the pMJ plasmids are shown in FIG. 1.

[0072] For insertion into the UL43 gene, plasmid p35 (kindly supplied byDr. C. A. MacLean; MacLean et al, 1991) was modified by insertion of anoligonucleotide linker, providing XbaI and XhoI sites, into the NsiIsite within the UL43 coding sequences. Plasmid pMJ101 was partiallydigested with XbaI, digested with XhoI and the larger fragment spanningthe β-gal gene plus HCMV enhancer was cloned between the XbaI and XhoIsites introduced into p35.

[0073] Plasmids were cleaved with ScaI and co-transfected into BHK cellswith in1814 DNA. Progeny viruses containing lacZ inserts were identifiedby the development of blue plaques in the presence of5-bromo-4-chloro-3indolyl-β-D galactopyranoside (X-gal) and purified bythree rounds of enrichment for lacZ-containing viruses. Final plaqueisolates were grown as small scale cultures and DNA was purified frominfected cells. For insertions in the TK gene, DNA was cleaved withEcoRI and analysed by Southern hybridization, using a ³² P radiolabelled2.4 kbp EcoRI fragment spanning the TK gene as probe. Insertion of lacZdisrupts the 2.4 kbp fragment, yielding species of 1.0 kbp and 2.0 or5.0 kbp, depending upon the structure of the lacZ-containing plasmidused. Virus isolates showing the correct pattern and lacking detectablehybridization to a 2.4 kbp fragment were grown as stocks.

[0074] For insertion into UL43, DNA was cleaved with EcoRI plus BamHIand probed with radiolabelled p35. Insertion of lacZ disrupts the 5.14kbp EcoRI/BamHI fragment, yielding smaller species. An isolate with theappropriate restriction pattern was grown as a virus stock.

[0075] Rescue of the insertion at the Vnw65 locus was achieved byco-transfecting pMC1 with DNA from lacZ-containing viruses. Viral DNAwas cleaved with BamHI, probed with pMC1 and isolates lacking a BamHIsite within the Vmw65 coding sequences were grown as virus stocks.

[0076] Virus titres were determined on BHK cells in the presence of 3 mMhexamethylene bisacetamide (McFarlane et al, 1992).

[0077] Inoculation of Mice

[0078] Viruses were inoculated subcutaneously into the right rearfootpad of 3 to 4 week old male BALB/c mice. Each mouse received 0.025ml containing approximately 8×10⁷ pfu of virus, diluted in ETC₁₀. Atvarious times after inoculation, lumbar dorsal root ganglia (DRG) fromspinal levels 2 to 6 (L2 to L6), and feet, were removed by dissectionand either used for reactivation studies, stored at −70° for virus assayor fixed in 4% formaldehyde at 4° for 1h for use in in situ β-gal assay.

[0079] β-gal Assays

[0080] For quantitative assay of β-gal levels, ganglia were homogenisedin phosphate buffered saline (PBS) and cells pelleted by centrifugation.Pellets were resuspended in 50 μl of lysis buffer (10 mM Tris/HCl,pH7.5; 2 mM MgCl₂; 10 mM NaCl; 0.1% Nonidet P40), maintained at 4° C. inlysis buffer for 5 min, mixed by vortexing and stored at −70° C. Afterthawing, 20 μl was assayed in a 100μl reaction mixture containing 25 mMTris/HCl, pH7.5; 125 mM NaCl; 2 mM MgCl₂; 13 mM β-mercaptoethanol and0.3 mM 4-methylumbelliferyl-β-D-galactoside. After incubation at 37° for2h, 3 μl 20% trichloroacetic acid was added. After 5 min at 4° C.,samples were centrifuged at 13,000 g for 5 min. Portions (100 μl ) ofthe supernatant were added to a reagent containing 133 mM glycine and 83mM Na₂CO₃, pH 10.7 and fluorescence measured in a TKO 100mini-fluorometer (Hoeffer). Protein concentrations of ganglion extractswere determined using a Sigma protein assay kit.

[0081] For in situ assay of β-gal, fixed DRG were washed twice with PBS,incubated at 37° C. in a mixture containing 5 mM potassium ferricyanide,5 mM potassium ferrocyanide, 2 mM MgCl₂, 0.02% Nonidet P40, 0.02% sodiumdeoxycholate and 1 mg/ml X-gal in PBS. Each reaction was performed on aglass slide in a volume of 150 μl, with a 22×40 mm coverslip sealed overthe DRG with wax. At 3, 24 or 48h the number or blue cells was countedand the DRG photographed using a Zeiss Axioskop microscope with MC80camera attachment. Sealed preparations were turned over to check thatβ-gal containing cells which could not be seen by focusing through thepreparation were not missed.

[0082] Reactivation DRG were excised and incubated for 3 days at 37° C.in ETC₁₀ plus 10% foetal calf serum and 2.5μg/ml amphotericin B,conditions previously used to induce reactivation of latent TK⁺ viruses(Ecob-Prince et al, 1993b; Ecob-Prince and Hassan, 1994). Explanted DRGwere then fixed and stained for the presence of β-gal.

[0083] Virus Assay

[0084] Feet or pooled L3/L4/L5 DRG were homogenized, frozen and thawedtwice, and sonicated prior to assay for virus, as described previously(Robertson et al., 1992).

[0085] In Situ Hybridization (ISH)

[0086] After the in situ assay for β-gal, the fixed explanted DRG fromeach mouse were embedded together in one block of paraffin,serially-sectioned and the percentage of neurons which expressed LATswas determined by ISH (Ecob-Prince et al, 1993a).

Example 1 Footpad Inoculation of in1853 and in1863

[0087] Mutants in1853 and in1863 were injected into mice via thefootpad. Their replication in the footpad and within the DRG wasmeasured and β-gal expression was examined in DRG at various times afterinoculation. The viruses possessed insertions of the lacZ genecontrolled by the HCMV enhancer, at the TK locus, and thus were expectednot to replicate in neurons. Injection of in1853 or in1863 at 8×10⁷ pfuresulted in no animal death and only a transient inflammation of thefootpad in approximately 10% of mice. High virus titres were observed inthe footpad at 2hr post infection (pi) of either in1853 or in1863 (FIG.2), but the amount of virus detected decreased over a period of 5 days,suggesting there was a failure to replicate at the periphery anddemonstrating that the absence of functional Vmw65 did not affect titresof these TK³¹ viruses in the foot. In contrast, the titres of 1814Rfollowing injection at 10₅ pfu per mouse were maintained within thefoot, as described previously (Robertson et al, 1992) When DRG wereexamined at 5 days post infection (pi), no virus (<5pfu in total) wasdetected in pooled L3/L4/L5/L6 ganglia of in1853-and in1863-infectedmice, whereas virus (90-4200 pfu) was detected in all animals infectedwith 1814R.

Example 2 Expression of β-gal in DRG Neurons

[0088] At various times after inoculation of mice or after explanationof latently-infected ganglia, individual DRG were scored by in situassay for the number of β-gal containing cells. Invariably, only L3, L4and L5 ganglia were positive, with the highest number usually in L4.Results are presented in Table 2 as a summation of the values from theseganglia, as suggested by Schmallbruch (1987). In preliminaryexperiments, DRG from 48 animals were scored after 3 or 21 h incubationin the β-gal reaction mixture, and the number found after 3h was, onaverage, 56% of that detected after 21h. No further increase wasobserved if the reaction time was extended to 48h, so counts werethereafter performed following incubation of ganglia for 24h.

[0089] The pattern of β-gal distribution in individual neurons varied,as observed by others (Ho and Mokarski, 1989; Dobson et al., 1990). Werecognised a heavy homogenous stain which often extended to the axon ofthe neuron, a pale homogenous pattern confined to the cell body, aspeckled distribution in which 5 to 20 foci of stain were observed ineach cell, and a pale-speckled pattern in which the foci weresuperimposed on a uniformly coloured background. To investigate therelationship between these patterns, DRG were photographed after 3h or21h in β-gal reaction mixture, and changes in the staining pattern ofindividual neurons observed. Some neurons initially classed as palebecame heavy, and some speckled became pale-speckled, but there waslittle evidence that cells with a speckled pattern at 3h becamehomogenously stained by 21h. The two basic patterns of staining,homogenous and speckled, thus appeared to represent distinctdistributions of enzyme rather than be the result of a simplequantitative difference.

[0090] The numbers of neurons in DRG infected with in1853 or in1863which contained β-gal were similar at either 3 or 5 days pi, with thehomogenous staining pattern predominating (Table 2). Very few neurons ofspeckled appearance were detected at 3 days pi, but cells of this typewere evident by 5 days. At 1 month pi, when latency was established, thenumber of β-gal containing neurons had fallen to about half of the valueat 3 days pi, and the staining was predominantly speckled (Table 2).However, this predominantly speckled pattern and the number of β-galcontaining cells was thereafter stable through 2 and 3 months pi and wasalso found in DRG at 5 months pi with in1853. These two characteristicsof β-gal expression did not appear to be influenced by the presence(in1863) or absence (in1853) of functional Vmw65 in the inoculatedvirus. The homogenous (usually heavy) pattern was found to predominateduring lytic replication (3 and 5 days pi) and reactivation followingexplantation, whereas the speckled pattern predominated at all times (1,2, 3 and 5 months pi) during latency.

[0091] To investigate whether the long-term detection of β-gal in micelatently-infected with in1853 and in1863 was due to specific features ofthe HCMV enhancer, additional virus mutants (Table 1) of the same basicdesign but with different promoters, regulating β-gal expression, in thepresence or absence of a nutated Vmw65 gene, were tested. Mutants in1852and in1862 contain the Momulv enhancer, whereas in1854 and in1864contained the IE Vmw65 gene promoter. These viruses were injected intomice and β-gal expression examined in DRG at 3 days and 1 month pi, andafter 3 months pi either directly or after explanation (Table 3). Inaddition, LAT-positive neurons were quantified by ISH at 3 months pi(Table 4). All mutants expressed β-gal at 3 days pi, with apreponderance of homogenously stained neurons. The numbers of positiveneurons were lower than found after infection with in1853 or in1863,particularly in the case of in1854 and in1864. No significant differencewas observed between viruses possessing Vmw65 mutations (in1852 andin1854) and their rescued progeny (in1862 and in1864 respectively).However, no β-gal containing cells were detected during latency afterinfection with these viruses or with in1855, but explantation resultedin the appearance of 10-20, mainly homogenously stained, neurons. Whenestablishment of latency was assessed by ISH, equivalent number ofLAT-expressing neurons were detected at 3 months post injection with allthe viruses (Table 4).

[0092] To investigate whether persistence of β-gal after infection within1853 or in1863 was due to the use of the TK gene as an insertion site,mutant in1891 (in which the UL43 gene was chosen as the insertion site)was constructed and tested. Mutants unable to express UL43 are able toestablish, maintain and reactivate from latency following injection ofmouse pinna (MacLean at el, 1991). At 3 days pi, in1891 caused anapparently more widespread infection of ganglia involving groups ofcells stained, thus counting of β-gal containing neurons at 3 days piwas not attempted. Nonetheless, during latency and reactivation, thepattern of β-gal expression was very similar to that observed afterinjection of in1853 (Table 5). The presence of enzyme was thus aconsequence of the use of the HCMV enhancer rather than the site ofinsertion or the TK⁻phenotype of in1853 and in1863.

[0093] The HCMV enhancer is one of the strongest known promoters, andvisualisation of stained ganglia suggested that in1853 and in1863expressed β-gal to greater extents than the other TK⁻viruses duringacute infection. To quantify enzyme levels, two groups of 9 mice wereinjected with in1853 or in1855, the most active mutant in lyticinfection based on visual observation of β-gal containing cells. At 3days pi L3, L4 and L5 DRG from the left (uninjected) or right (injected)sides of each of 6 animals were removed by dissection and assayedindividually for β-gal levels. The average levels of activity found inDRG of the left side of each group were remarkably similar (Table 6).Compared to these background levels, DRG from mice infected with in1853were 6.6 times higher, and those from mice infected with in1855 were 1.8times higher, than background An in situ β-gal assay of DRG from 3 othermice for each virus yielded an average of 122 (for in1853) and 66 (forin1855) stained neurons per mouse, similar to the values obtainedpreviously (Tables 2, 3 and 5), confirming that (even allowing for thedifference in numbers of β-gal positive cells) in1853 expressed moreβ-gal than in1855.

[0094] The results present here demonstrate that the presence or absenceof functional Vmw65 in the virus did not alter any aspect ofheterologous lacZ gene expression or of latency. Viruses which wereVmw65 negative (in1852, in1853 and in1854) could not be distinguishedfrom those which were Vmw65 positive (in1862, in1863 and in1864) in thisrespect.

[0095] Those mutant viruses which contained the HCMV enhancer (in1853,in1863 and in1891) showed good long-term expression of the insertedheterologous lacZ marker gene during latency. However in1863 and in1891being Vmw65 positive and TK positive respectively are virulent and thusunsuitable for use as a viral vector in vivo. TABLE 1 Characteristics ofthe viruses used. Promoter for Location of Virus lacZ constructPhenotype * in 1853 HCMV TK TK⁻/Vmw65⁻ in 1863 HCMV TK TK⁻/Vmw65⁺ in1852 Momulv TK TK⁻/Vmw65⁻ in 1862 Momulv TK TK⁻/Vmw65⁺ in 1854 Vmw110 TKTK⁻/Vmw65⁻ in 1864 Vmw110 TK TK⁻/Vmw65⁺ in 1855 Vmw65 TK TK⁻/Vmw65⁻ in1891 HCMV UL43 TK⁺/Vmw65⁻

[0096] TABLE 2 The proportions of cells positive for B-gal which havedifferent patterns of staining at different times after infection ofexplantation of the DRG. Time after Total* infection or (L3, 4, Pale/explantation 5) Heavy Pale Speckled Speckled in 1853: 3 dpi 103  82(79%) 10 (9%)  8 (8%)  3 (3%) 5 dpi 88 21 (24%) 36 (41%) 22 (25%)  9(10%) 1 mpi 37  3 (8%)  3 (7%) 27 (74%)  4 (11%) 2 mpi 65  9 (14%) 16(25%) 28 (43%) 12 (18%) 3 mpi 49  3 (6%)  2 (4%) 38 (78%)  6 (12%) 5 mpi66  1 (1%)  0 55 (86%) 10 (13%) 3 dpe 78 29 (37%) 32 (40%) 14 (18%)  4(5%) in 1863: 3 dpi 70 44 (63%) 10 (14%) 12 (17%)  4 (6%) 5 dpi 65 12(18%) 25 (39%) 22 (33%)  6 (10%) 1 mpi 49  3 (7%)  1 (3%) 40 (82%)  4(8%) 2 mpi 51  2 (4%)  4 (6%) 43 (84%)  3 (6%) 3 mpi 57  3 (6%)  6 (10%)37 (65%) 11 (19%) 3 dpe 78 28 (36%) 31 (40%) 16 (20%)  3 (4%)

[0097] TABLE 3 The proportions of cells positive for b-gal which showdiferent patterns of staining at different times after infection orexplantation of the DRG infected with viruses containing lacZ controlledby different promoters in the TK gene location. Time after Total*infection or (L3, 4, Pale/ explantation 5) Heavy Pale Speckled speckled3 dpi: in 1862 41  9 (22%) 23 (56%)  8 (20%) 1 (2%) in 1852 57  9 (15%)33 (58%) 12 (21%) 3 (6%) in 1864 18  5 (25%) 13 (75%) zero zero in 185422  6 (27%) 16 (73%) zero zero in 1855 77 22 (28%) 54 (70%)  1 (2%) zero1 mpi zero zero zero zero zero All viruses 3 mpi zero zero zero zerozero All viruses 3 dpe in 1862 11  1 (9%) 10 (91%) zero zero in 1852 12 3 (25%)  7 (50%)  1 (8%) 1 (9%) in 1864 10  2 (20%)  7 (70%)  1 (10%)zero in 1854 16  4 (25%) 12 (75%) zero zero in 1855 17  2 (12%) 15 (88%)zero zero

[0098] TABLE 4 A comparison of the total number of neurons in L3, L4 andL5 DRG which contained either β-gal or LATs at 3 months pi. Virus β-galpositive neurons LAT-positive neurons in 1862 0 192 in 1852 0 161 in1864 0 117 in 1854 0 155 in 1853 49  143 in 1863 57  218 in 1891 32  172in 1855 0 224

[0099] TABLE 5 The proportions of cells positive for β-gal which showeddifferent patterns of staining at different times after infection orfollowing explantation of the DRG infected with viruses containingHCMV-lacZ in different locations. Time after Total infection or (L3, 4,Pale/ explantation 5) Heavy Pale Speckled speckled in 1853: 3 dpi 110*35 (32%) 65 (59%)  9 (8%) 1 (1%) 1 mpi  23* zero  1 (3%) 17 (77%) 5(20%) 3 mpi  43† zero  3 (8%) 38 (89%) 2 (3%) 3 dpe  75† 30 (40%) 32(42%)  8 (10%) 6 (8%) in 1891 1 mpi  15* <1 (4%)  3 (19%) 10 (69%) 1(8%) 3 mpi  32† zero  4 (13%) 21 (65%) 7 (22%) 3 dpe  67† 29 (42%) 30(44%)  8 (13%) zero

[0100] TABLE 6 Assay for β-gal in DRG 3 days pi with different virusconstructs. Animal Units of β-gal per μg protein Virus no. Left* Right*Right/Left in 1853 1. 191 1562 7.7 2. 325 1767 5.4 3. 350 2828 8.1 4.196 1161 5.9 5. 219 1238 5.7 6. 126  858 6.8 average 235 1569# 6.6 in1855 7. 212  322 1.5 8. 426  869 2.0 9. 445  653 1.5 10.  157  223 1.411.  107  307 2.9 12.  119  174 1.5 average 244  425## 1.8

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1. A recombinant herpes simplex virus (HSV) viral vector genome whichcomprises; (i) a DNA sequence change in the gene coding for Vmw65protein such as to substantially remove transinducing propertiesthereof; and (ii) an expressable heterologous gene inserted into aregion of the HSV genome which is non-essential for culture of thevirus, the gene being under the control of the immediate early 1 (IE1)gene enhancer of cytomegalovirus (CMV).
 2. A recombinant HSV genomeaccording to claim 1 wherein the CMV gene enhancer is the humanenhancer.
 3. A recombinant HSV genome according to claim 1 wherein theCMV gene enhancer contains sequence variations such that the enhancerproperties are not affected.
 4. A recombinant HSV genome according toany preceding claim which is derived from HSV-1.
 5. A recombinant HSVgenome according to any preceding claim wherein the sequence change inthe gene coding for Vmw65 is an insertion.
 6. A recombinant HSV genomeaccording to claim 5 wherein the insertion is of 3-72 base pairs.
 7. Arecombinant HSV genome according to any preceding claim wherein thesequence change in the gene coding for Vmw65 protein is at a positioncorresponding to between amino acids 289 and 480 of the protein.
 8. Arecombinant HSV genome according to any preceding claim wherein theinserted expressable heterologous gene is selected from (a) human, rator mouse tyrosine hydroxylase genes 1, 2 or 3, which are relevant to thealleviation of symptoms of Parkinson's disease; (b) human, rat or mousenerve growth factor (e.g. the beta subunit) for treatment of Alzheimer'sdisease and Parkinson's disease; (c) human, rat or mousehypoxanthine-guanine phosphoribosyl transferase gene for the treatmentof Lesch-Nyhan disease; and (d) human beta-hexosaminidase alpha chaingene, for the treatment of Tay-Sachs and Sandhoff's diseases; and (e)human immunodeficiency virus (HIV) nef gene, for the control ofneurological symptoms in HIV-positive individuals.
 9. A recombinant HSVgenome according to any preceding claim wherein the heterologous gene isinserted in the coding sequences or in the flanking control regions ofone or more of the following HSV-1 genes:
 1. The UL23 gene
 2. The RL1gene
 3. The RL2 gene
 4. The locus encoding the latency associatedtranscripts.
 5. The UL2 gene
 6. The UL3 gene
 7. The UL4 gene
 8. The UL10gene
 9. The UL11 gene
 10. The UL13 gene
 11. The UL16 gene
 12. The UL20gene
 13. The UL24 gene
 14. The UL40 gene
 15. The UL41 gene
 16. The UL43gene
 17. The UL44 gene
 18. The UL45 gene
 19. The UL46 gene
 20. The UL47gene
 21. The UL50 gene
 22. The UL55 gene
 23. The UL56 gene
 24. The US1gene
 25. The US2 gene
 26. The US3 gene
 27. The US4 gene
 28. The US5 gene29. The US7 gene
 30. The US8 gene
 31. The US9 gene
 32. The US10 gene 33.The US11 gene
 34. The US12 gene
 10. An HSV mutant containing the genomeof any preceding claim.
 11. Use of the recombinant HSV genome of any ofclaims 1 to 9 or the HSV mutant of claim 10 for disease therapy.
 12. Useof the recombinant HSV genome of any of claims 1 to 9 or the HSV mutantof claim 10 as a vaccine.
 13. A pharmaceutical composition foradministering the viral vector genome, which comprises the recombinantHSV genome of any of claims 1 to 9 or the HSV mutant of claim 10 inadmixture with a pharmaceutically acceptable carrier.